Advanced Histological Techniques
Staining Tissue Sections for Electron Microscopy
Although secondary fixation in osmium tetroxide provides some areas of electron density, this is usually not sufficient to provide high contrast, high definition images. A number of staining techniques are available to enhance the contrast of areas of interest. These fall into two major categories. Positive stains deposit electron dense material on the area of interest, so that it stands out as a dark area on a light background. Negative stains penetrate and darken the interstices between areas of interest, which then appear light on a dark background.
Uranyl acetate is used as a positive stain for EM. Uranyl ions react strongly with phosphate and amino groups, staining DNA and some proteins. Organelles composed of membranes are not stained well. Note that the starting material is radioactive. Lead citrate may also be employed as a positive stain. Reynolds lead citrate stain binds lead ions to negative ions, producing a general increase in contrast. Lead is a cumulative toxin, so skin contact must be avoided.
Negative staining is most often used to highlight surface features on individual particles, such as virions, bacteria, or cell fragments.
Antibodies bound to colloidal gold particles are visible under the EM as dark spots. The gold particles can range from 1 to 20 nm, although 5-10 nm seems to be an optimum range. Embedded or frozen sections can be probed in this way.